Factor XII and uPAR upregulate neutrophil functions to influence wound healing

Abstract

Coagulation factor XII (FXII) deficiency is associated with decreased neutrophil migration, but the mechanisms remain uncharacterized. Here, we examine how FXII contributes to the inflammatory response. In 2 models of sterile inflammation, FXII-deficient mice (F12-/-) had fewer neutrophils recruited than WT mice. We discovered that neutrophils produced a pool of FXII that is functionally distinct from hepatic-derived FXII and contributes to neutrophil trafficking at sites of inflammation. FXII signals in neutrophils through urokinase plasminogen activator receptor-mediated (uPAR-mediated) Akt2 phosphorylation at S474 (pAktS474). Downstream of pAkt2S474, FXII stimulation of neutrophils upregulated surface expression of αMβ2 integrin, increased intracellular calcium, and promoted extracellular DNA release. The sum of these activities contributed to neutrophil cell adhesion, migration, and release of neutrophil extracellular traps in a process called NETosis. Decreased neutrophil signaling in F12-/- mice resulted in less inflammation and faster wound healing. Targeting hepatic F12 with siRNA did not affect neutrophil migration, whereas WT BM transplanted into F12-/- hosts was sufficient to correct the neutrophil migration defect in F12-/- mice and restore wound inflammation. Importantly, these activities were a zymogen FXII function and independent of FXIIa and contact activation, highlighting that FXII has a sophisticated role in vivo that has not been previously appreciated.

Keywords: Cell Biology; Inflammation; Neutrophils.

Figure 1. FXII influences leukocyte migration into skin wounds.

(A and C) Frozen sections of day 2 (D2) and day 5 (D5) wounds were stained with anti-CD11b antibody to assess leukocyte infiltration. (B and D) Total number of CD11b cells per high-power field (HPF). WT, n = 10; F12^–/–, n = 10 mice (B); WT, n = 8; F12^–/–, n = 10 mice (D). (E and G) Representative frozen sections from day 2 and day 5 wounds were stained with anti-Ly6G antibody to determine neutrophil infiltration. (F and H) The numbers of Ly6G cells/high-power field are shown. WT, n = 10; F12^–/–, n = 10 mice (F); WT, n = 8; F12^–/–, n = 10 mice (H). Fluorescent images were obtained using a Nikon TE2000-S microscope at ×20 magnification. CD11b and Ly6G staining in all panels was compared by morphometric analysis of cell number per high-power field using ImageJ software (NIH). Data represent mean ± SEM. *P < 0.01 vs. WT control mice by Student’s t test. Scale bar: 50 μm.

Figure 2. Leukocyte migration in TG-induced peritonitis.

WT and F12^–/– mice were injected intraperitoneally with TG solution. At 4 hours (A), mice were subjected to peritoneal lavage and the PEC number was determined. WT, n = 7; F12^–/–, n = 11 mice (mean ± SEM). *P = 0.0001 vs. WT control mice by Student’s t test. (B) Giemsa-Wright stain of peritoneal lavage fluid. Original magnification, ×4. Scale bar: 10 μm. Representative images of n = 5 WT and n = 7 F12^–/– animals. (C) Quantitation of neutrophil population in the peritoneal lavage fluid of WT and F12^–/– mice 4 hours after TG instillation as observed by light microscopy (WT, n = 5; F12^–/–, n = 7). Data represent mean ± SEM. *P < 0.0001 vs. WT control mice by Student’s t test. (D) Murine peritoneal lavage fluid was labeled with PE-conjugated anti-CD11b and PerCPCy 5.5–conjugated anti–F4-80 antibodies. Circles indicate the neutrophil subpopulation (CD11b-positive, F4-80–negative cells). These figures are representative of flow cytograms of n = 4 animals in each group.

Figure 3. Influence of FXII on wound healing.

(A) Full thickness wounds from WT and F12^–/– mice (n = 12 mice/group) were imaged daily until closure using a Nikon SMZ-U dissecting microscope. Original magnification, ×1. (B) The wound area (mm²) was measured daily from day 0 until closure in WT (n = 20) and F12^–/– (n = 28) mice. Wounds were considered closed when their areas relative to day 0 were less than 5%. Mean ± SEM. *P < 0.05, 2-way ANOVA. (C) H&E-stained sections from day 5 were analyzed to determine the degree of reepithelialization. Black line demarcates the total length of wound space; yellow line represents the remaining wound gap. Red arrows indicate the epithelial tongues showing ingrowing epithelium. All histologic sections were obtained using a Leica SCN 400 slide scanner equipped with a Hamamatsu line sensor color camera at ×4 magnification. Scale bar: 100 μm. (D) The percentage of reepithelialization (% wound closure) was determined using the ImageJ software and is shown for WT (n = 16) and F12^–/– (n = 10) mice. Mean ± SEM. *P < 0.05, Student’s t test. (E and G) Sections from day 2 wounds were stained for NE (E) at ×20 magnification or H3Cit (G) at ×10 magnification, respectively. Scale bars: 50 μm (E); 20 μm (G). (F) NE content was determined in the wound bed immediately beneath the scab in day 2 wounds and presented as percentage of NE-stained cells to total number of cells per high-power field. WT, n = 6; F12^–/–, n = 7. Mean ± SEM. *P = 0.001, Student’s t test. (H) H3Cit content of day 2 wounds is presented as total number of cells per high-power field. WT, n = 8; F12^–/–, n = 15. Mean ± SEM. *P < 0.04, Student’s t test.

Figure 4. Influence of F12 siRNA on inflammation and thrombosis.

(A) Plasma FXII coagulant activity following siRNA treatment. WT mice (n = 6/time point) were treated by tail-vein injection with F12 or luciferase siRNA at 0.1 mg/kg dose. At indicated times, blood was collected and residual plasma FXII activity was determined by an aPTT-based assay. (B) Western blot for FXII was performed under reduced conditions 8 hours and 24 hours after F12 siRNA treatment. Representative blot of n = 4. (C) WT mice were treated intravenously with luciferase (Luc siRNA) or F12 siRNA. Twenty-four hours after dosing, liver was collected for hepatic mRNA expression. Note the specificity of F12 siRNA for F12 gene silencing. Mean ± SEM. n = 5 individual experiments run in triplicate. *P = 0.001 by mean row statistics. (D and E) Twenty-four hours after siRNA treatment, mice received 5-mm full-thickness punch biopsies. Wounds were harvested on day 2 and day 5, and frozen sections were stained with anti-Ly6G antibody. (D) Number of Ly6G cells/HPD in day 2 wounds. WT, n = 6; F12^–/–, n = 10; Luc siRNA treated, n = 10; F12 siRNA treated, n = 9. Mean ± SEM. *P < 0.006, 1-way ANOVA. (E) Number of Ly6G cells/high-power field in day 5 skin wounds. WT, n = 7; F12^–/–, n = 6; Luc siRNA treated, n = 7; F12 siRNA treated, n = 8. Mean ± SEM. *P < 0.02, 1-way ANOVA. (F) WT mice were treated with F12 siRNA (n = 8) or luciferase siRNA (n = 7), and 24 hours later, they underwent TG-induced peritonitis. PEC number was determined at 4 hours. Mean ± SEM. P = 0.837 by Student’s t test. (G) PEC number 4 hours after TG instillation in WT (n = 10), F12^–/– (n = 8), and F12^–/– mice reconstituted with purified human FXII (n = 3) or recombinant mouse FXII (mFXII) (n = 9) to physiologic plasma FXII levels of 450–650 nM. Mean ± SEM. *P ≤ 0.0003 vs. WT, 1-way ANOVA.

Figure 5. Neutrophils (PMNs) express F12 mRNA and FXII.

(A) Left panel: murine total mRNA was isolated from BM-derived neutrophils (PMNs 1,2) or homogenized livers (Liver 1–3), and first-strand cDNA was synthesized with SuperScript III reverse transcriptase. The PCR product from the same animal on an exon 1–6 probe is shown. Images are representative of 3 experiments. Right panel: relative F12 expression in liver and BM-derived neutrophils as determined by 2^–ΔΔCt. Mean ± SEM. *P = 0.0001, Student’s t test. (B) Isolated WT (left panel) and F12^–/– (right panel) murine peripheral blood neutrophils were incubated with media or fMLP for 2 hours. Cells were stained with primary antibody directed against FXII (green); nuclei were counterstained with DAPI (blue). Images are representative of n = 3 experiments. Original magnification, ×20. Scale bar: 10 μm. (C) Confocal visualization of FXII in normal human peripheral neutrophils. Neutrophils were incubated with media (UT; top panels) or fMLP (bottom panels), then either not permeabilized (surface) or permeabilized (intracellular) and stained with antibody to FXII. Confocal microscopy indicates the intracellular location of FXII protein before treatment and trafficking to the cell surface after stimulation with fMLP. Images shown are representative of 3 individual experiments. Original magnification, ×20 (left panel); ×100 (right panel). Scale bar: 5 μm. (D) Confocal FXII visualization in neutrophils from FXII-deficient patient in UT (top) and fMLP-treated (bottom) cells. FXII is absent in peripheral neutrophils from a patient with congenital FXII deficiency. Original magnification, ×10. Scale bar: 10 μm.

Figure 6. FXII is secreted from neutrophils and signals through uPAR to promote pAktS⁴⁷³ and pAktS⁴⁷⁴.

(A) WT neutrophils (PMNs) were incubated in the absence or presence of fMLP. At indicated time points, cells and supernatant were separated and resolved on SDS-PAGE. FXII was detected with anti-FXII antibody. Representative blot of n = 3. (B) Sensorgram of FXII binding to immobilized uPAR in the presence (solid lines) or absence (dotted lines) of 10 μM Zn²⁺. Increasing concentrations of FXII (0 nM, 1 nM, 10 nM, 100 nM, 400 nM) were injected over a uPAR-immobilized, gelatin-coated CM5 chip. Each sensorgram line represents an average of 3 injection runs as described in Methods. Analyte injection was terminated at 120 seconds. (C–E, top panels) PMNs were treated with fMLP, FXII/Zn²⁺, or Zn²⁺ alone for 5 minutes, followed by immunoblotting. Wort or LY lanes were pretreated with Wortmannin (50 μM) and LY294002 (100 μM) for 1 hour, respectively, followed by FXII/Zn²⁺ treatment. (C–E, bottom panels) Percentage pAktS⁴⁷³ in neutrophils. (F–H, top panels) Neutrophils were treated with fMLP, FXII/Zn²⁺, or FXII alone. Lanes labeled Akti were pretreated with Akti XII (5 μM) for 30 minutes, followed by FXII/Zn²⁺ treatment. Where indicated, cells were pretreated with TPEN (10 μM) for 30 minutes before stimulation with fMLP or FXII/Zn²⁺. Lysates were immunoblotted with antibodies against pAkt2S⁴⁷⁴. (F–H, bottom panels) Percentage of pAkt2S⁴⁷⁴ (% relative density units [RDU]) in neutrophils. UT cell band density was considered 0%; band density of fMLP-treated cells subtracted from UT band density was set at 100%. Data in panels D–I represent mean ± SEM of 4 or more experiments. *P < 0.0001, 1-way ANOVA.

Figure 7. FXII signaling in neutrophils is a zymogen function.

(A) WT neutrophils were incubated in the absence (UT) or presence of 62 nM FXII and 10 μM Zn²⁺ for 1, 2, and 5 minutes. Western blot analysis for FXII was performed under reduced conditions using polyclonal anti-FXII antibody. Representative blot of n = 3. (B) Chromogenic assay of FXIIa activity. Neutrophils were allowed to adhere on a gelatin-coated 96-well plate for 10 minutes and subsequently incubated with fMLP, 62 nM FXII/10 μM Zn²⁺, or rising concentrations of FXIIa (0.62 nM, 6.2 nM, 62 nM)/10 μM Zn²⁺. S-2302 (200 μM) was added in each well, and changes in OD₄₀₅ were continuously monitored on a microplate reader. Graph represents mean OD of a single experiment run in triplicate. (C) Left panel: cleavage of S-2302 is presented as change in mean OD (ΔOD) over the first 5 minutes of neutrophil incubation with media, fMLP, FXII/Zn²⁺, or rising concentrations of FXIIa/Zn²⁺. Note that 5 minutes is the time point used in pAkt2S⁴⁷⁴ signaling studies. Right panel: cleavage of S-2302 over 180 minutes. Graphs are presented in box-and-whiskers diagrams where minimum/maximum distribution and outliers are shown. Mean ΔOD, n = 4 individual experiments run in triplicate. (D) FXII immunoblotting of supernatant from HEK293 cells expressing WT FXII, FXII Locarno (FXII-R353P), and FXII double-mutant (FXII-D) (combined R353P and S544 substitutions). Mock: supernatant from nontransfected cells; Purified FXII: plasma-derived mouse FXII. (E) Neutrophils were treated with WT FXII/ Zn²⁺ or FXII-D variant and Zn²⁺. Lanes labeled Akti were pretreated with Akti XII (5 μM) for 30 minutes, followed by FXII/Zn²⁺ or FXII-D/Zn²⁺. Lysates were immunoblotted with antibodies against pAkt2S⁴⁷⁴. Media: conditioned media from transfected cells. (F) Percentage of pAkt2S⁴⁷⁴ (% RDU) in neutrophils. Mean ± SEM, n = 4 experiments. *P < 0.0001, 1-way ANOVA.

Figure 8. The FXII-uPAR axis promotes neutrophil adhesion and chemotaxis.

(A) WT (n = 7) and F12^–/– (n = 5) peripheral neutrophils (1 × 10⁶/ml) were incubated with fMLP and applied on BSA- or fibrinogen-coated 96-well plates. Adhered cells were determined by a fluorogenic assay reported in relative fluorescence units (RFU), 480/520 nm. Mean ± SEM. *P < 0.02 vs. WT neutrophils by Student’s t test. (B) Boyden chamber chemotaxis. Medium alone, fMLP or 62 nM FXII, and 10 μM Zn²⁺ were placed in the lower wells of a Boyden chamber. WT (n = 11) and F12^–/– (n = 12) neutrophils (1 × 10⁶/ml) were placed in the upper wells. Chemotaxis was determined by measuring fluorescence in the lower wells. Mean ± SEM vs. fMLP and FXII/Zn²⁺ treatment(s). *P < 0.0001, 1-way ANOVA with Bonferroni’s correction. (C–H) Microfluidic channel assay of live-cell chemotaxis. Murine neutrophils (1 × 10⁶/ml) were applied onto Matrigel-coated microfluidic channels that contained an interphase with a pocket infused with 62 nM FXII and 10 μM Zn²⁺, 10 μM fMLP, or media. Images at the interphase were obtained every 5 minutes for 2 hours. WT (C), F12^–/– (E), and Plaur^–/– (G). Cumulative linear regression curves of relative cell increase of neutrophils at the interphase over 2 hours. WT (D), F12^–/– (F), and Plaur^–/– (H). Chemotaxis rates of neutrophils from each indicated genotype as determined by the slope of linear regression curves. Graphs plotted as mean ± SEM, n = 3–6 experiments. P values in each panel were determined by 1-way ANOVA with Bonferroni’s correction.

Figure 9. FXII signaling in neutrophils regulates α_Mβ₂ surface expression, intracellular Ca²⁺ mobilization, and NET formation.

(A) Surface expression of α_Mβ₂ integrin on UT (grey curve) and FXII/Zn²⁺–stimulated (pink curve) neutrophils. Representative flow diagram of n = 7 individual experiments. (B) Quantitation of α_Mβ₂ antibody binding (mean fluorescent intensity [MFI]). Mean ± SEM, n = 7 experiments. *P < 0.03 UT vs. FXII/Zn²⁺, 1-way ANOVA. (C) WT neutrophils were loaded with 1 μM Fluro-4-AM Ca²⁺ dye for 45 minutes and then treated with or without 5 mM ATP, 3 μM ionomycin, 10 μM fMLP, FXII (62 nM or 100 nM)/10 μM Zn²⁺, or Zn²⁺ alone. Intracellular Ca²⁺ mobilization was measured at 30-second intervals for 30 minutes. (D) Intracellular Ca²⁺ concentration with various agonists. Mean ± SEM, n = 4, each run in triplicate. *P < 0.0001 vs. UT cells, 1-way ANOVA. (E) WT neutrophils were stimulated with fMLP, 100 nM PMA, or FXII/Zn²⁺ for 120 minutes, and immunoblot analysis was performed for H3-C. Representative blot of n = 4. (F) H3-C fold increase compared with UT neutrophils. Mean ± SEM, n = 4. *P < 0.007 UT vs. FXII/Zn²⁺, 1-way ANOVA. (G) WT neutrophils were stimulated with 62 nM FXII/10 μM Zn²⁺ for the indicated times. Immunoblot analysis was performed for H3-C. Representative blot of n = 4. (H) H3-C fold increase compared with UT neutrophils. Mean ± SEM, n = 4. *P = 0.007 at 30 minutes; **P < 0.002 at 60 minutes; ***P = 0.03 at 120 minutes vs. UT, 1-way ANOVA. (I) NETotic index rate of WT neutrophils activated with fMLP, 4 μM A23187, 1 μM PMA, FXII/Zn²⁺, or Zn²⁺ alone. Where indicated, neutrophils were preincubated with 5 μM Akti XII or 300 μM LRG20 for 30 minutes before stimulation with FXII/ Zn²⁺. Curves represent mean ± SEM except A23187 (shown as mean). n = 5–9 each run in triplicate. *P < 0.0001 FXII/ Zn²⁺ vs. media at 150–400 minutes, 2-way ANOVA.

Figure 10. The influence of BM transplantation on inflammation and wound healing.

(A) PEC number after TG-induced peritonitis in WT or KO (F12^–/–) hosts after WT or KO BM transplantation (n = 11–13 transplants/condition). Mean ± SEM. *P < 0.0004 by 1-way ANOVA. (B) Flow cytometry of murine peritoneal lavage fluid 4 hours following TG-induced peritonitis in WT or KO BM–transplanted hosts. PEC were labeled with PE-conjugated anti-CD11b and PerCPCy 5.5–conjugated anti–F4-80 antibodies. Circles indicate the neutrophil subpopulation (CD11b-positive, F4-80–negative cells). The figure shows a representative flow cytogram of n = 4 transplanted animals in each group. (C) NE and pAkt coimmunostaining from day 2 wounds of WT and (D) day 2 wounds of F12^–/– BM chimeras. Representative images of n = 4 wounds in each group. Left panels in C and D. Scale bars: 50 μm (left panels); 100 μm (right panels). Original magnification, ×4 (left panels); ×20 (right panels). (E) Number of Ly6G cells/high-power field in day 2 wounds of WT and F12^–/– chimeras. n = 6–9 transplants/condition. Mean ± SEM. *P < 0.007, 1-way ANOVA. (F) H&E-stained sections in day 5 wounds of WT and F12^–/– BM chimeras. Representative images of n = 6–9 wounds in each group of transplanted animals. Original magnification, ×4. Black lines demarcate the total length of original wound; yellow line represents the remaining wound gap. Red arrows represent the ingrowing epithelial tongues, closing wound. In photographs in which a red asterisk is seen, the wound gap is 0, indicating that the wound has completely reepithelialized. Scale bars: 100 μm. Histologic sections were obtained using a Leica SCN 400 slide scanner equipped with a Hamamatsu line sensor color camera. Analysis was performed using ImageJ software. (G) Wound closure in WT and F12^–/– BM chimeras, n = 6–9 wounds/group. Mean ± SEM. *P = 0.0004, 1-way ANOVA.

Figure 11. Role of FXII in inflammation and wound healing.

Neutrophil FXII functions as an autocrine messenger upstream of plasma FXII. FXII signals through uPAR to promote AktS⁴⁷³ and Akt2S⁴⁷⁴ phosphorylation. Propagation of FXII-mediated neutrophil activities includes adhesion, migration, chemotaxis that leads to neutrophil trafficking at sites of inflammation, and NET formation. Activities of neutrophil-derived FXII are distinct from the function of plasma-derived FXII, which mediates contact activation on the surface of preformed NETs to produce FXIIa for blood coagulation.